Carboxypeptidase A and

Metabolic Functions. Zinc is essential for the function of many enzymes, either in the active site, ie, as a nondialyzable component, of numerous metahoenzymes or as a dialyzable activator in various other enzyme systems (91,92). WeU-characterized zinc metahoenzymes are the carboxypeptidases A and B, thermolysin, neutral protease, leucine amino peptidase, carbonic anhydrase, alkaline phosphatase, aldolase (yeast), alcohol... 

ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use A, B, C, and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators. 

The body of an adult human contains about 2 g of Zn but, as Zn enzymes are present in most body cells, its concentration is very low and realization of its importance was therefore delayed. The two Zn enzymes which have received most attention are carboxypeptidase A and carbonic anhydrase. 

Although zinc, cadmium, and mercury are not members of the so-called main-group elements, their behavior is very similar because of their having complete d orbitals that are not normally used in bonding. By having the filled s orbital outside the closed d shell, they resemble the group IIA elements. Zinc is an essential trace element that plays a role in the function of carboxypeptidase A and carbonic anhydrase enzymes. The first of these enzymes is a catalyst for the hydrolysis of proteins, whereas the second is a catalyst for the equilibrium involving carbon dioxide and carbonate,... 

One of the most important metals with regard to its role in enzyme chemistry is zinc. There are several significant enzymes that contain the metal, among which are carboxypeptidase A and B, alkaline phosphatase, alcohol dehydrogenase, aldolase, and carbonic anhydrase. Although most of these enzymes are involved in catalyzing biochemical reactions, carbonic anhydrase is involved in a process that is inorganic in nature. That reaction can be shown as... 

Figure 12.4 Active sites of thermolysin and carboxypeptidases A and B. (Reprinted with permission from Parkin, 2004. Copyright (2004) American Chemical Society.)...

Fig. 3.11. Simplified representation of the complex between carboxypeptidase A and glycyl-...

Proteins crystallized from very low salt concentrations (examples are carboxypeptidase A and elastase) can often be treated exacdy like proteins crystallized from alcohol-water mixtures. Their low solubility in water allows them to be transferred from their normal mother liquor to a distilled water solution or to a solution of low (10-20%) alcohol concentration without disorder. It is advisable to carry out this transfer at near 0 C to further decrease the protein solubility. From this stage it is trivial to add alcohol while cooling, as described above. Complications arise, however, when the salt employed as a precipitant in the native mother liquor is insoluble in alcohols. The solution to this problem is to replace the salt by ammonium acetate at equivalent or higher ionic strength. Ammonium acetate is soluble up to 1 M in pure methanol, and is very soluble in nearly all alcohol-water mixtures, even at low temperature. It therefore provides a convenient substitute for salts such as sodium sulfate or sodium phosphate. 

Zinc is a microelement essential for proper functioning of the human body. The level of daily demand for zinc was established as 13 to 16 mg (Ziemlahski, 2001). Zinc plays a role in protein and carbohydrate metabolism and is a component of over 60 metaloenzymes, including alkaline phosphatase, pancreatic carboxypeptidases A and B, alcoholic and lactic dehydrogenases, carbonate anhydrase, and proteases. It also forms bonds with nucleic acids -which is very important for their functioning (Prasad, 1983 Valee and Falchuk, 1993). 

A novel concept of using bioadhesive polymers as enzyme inhibitors has been developed [97]. Included are derivatives of poly acrylic acid, polycarbophil, and car-bomer to protect therapeutically important proteins and peptides from proteolytic activity of enzymes, endopeptidases (trypsin and a-chymotrypsin), exopeptidases (carboxypeptidases A and B), and microsomal and cytosolic leucine aminopeptidase. However, cysteine protease (pyroglutamyl aminopeptidase) is not inhibited by polycarbophil and carbomer [97]. 

The three hydrolytic enzymes that have been discussed, a-chymo-trypsin, carboxypeptidase A, and lysozyme, cover a wide range of substrate types and mechanistic possibilities. Formulation of principles which might apply to enzymatic catalysis in general is difficult from such a small sampling, but certain features of the enzymatic and model reactions warrant some comment. 

Zinc proteases carboxypeptidase A and thermolysin have been extensively studied in solution and in the crystal (for reviews, see Matthews, 1988 Christianson and Lipscomb, 1989). Both carboxypeptidase A and thermolysin hydrolyze the amide bond of polypeptide substrates, and each enzyme displays specificity toward substrates with large hydrophobic Pi side chains such as phenylalanine or leucine. The exopeptidase carboxypeptidase A has a molecular weight of about 35K and the structure of the native enzyme has been determined at 1.54 A resolution (Rees et ai, 1983). Residues in the active site which are important for catalysis are Glu-270, Arg-127, (liganded by His-69, His-196, and Glu-72 in bidentate fashion), and the zinc-bound water molecule (Fig. 30). 

Carboxypeptidase A was the first zinc enzyme to yield a three-dimensional structure to the X-ray crystallographic method, and the structure of an enzyme-pseudosubstrate complex provided a model for a precatalytic zinc-carbonyl interaction (Lipscomb etai, 1968). Comparative studies have been performed between carboxypeptidase A and thermolysin based on the results of X-ray crystallographic experiments (Argosetai, 1978 Kesterand Matthews, 1977 Monzingoand Matthews, 1984 Matthews, 1988 Christianson and Lipscomb, 1988b). Models of peptide-metal interaction have recently been utilized in studies of metal ion participation in hydrolysis (see e.g., Schepartz and Breslow, 1987). In these examples a dipole-ion interaction is achieved by virtue of a chelate interaction involving the labile carbonyl and some other Lewis base (e.g.. 

The collapse of the proteolytic tetrahedral intermediate of the promoted-water pathway requires a proton donor in order to facilitate the departure of the leaving amino group. Rees and Lipscomb (1982) considered Glu-270, but favored Tyr-248 for this role, but Monzingo and Matthews (1984) fully elaborated on a role for Glu-270 of carboxypeptidase A and Glu-143 of thermolysin as intermediate proton donors. This proposal for carboxypeptidase A is corroborated by the near-normal activity observed for the Tyr-248- Phe mutant of rat carboxypeptidase A (Garden et al, 1985 Hilvert et al, 1986) and is reflected in the mechanistic scheme of Fig. 31 (Christianson and Lipscomb, 1989). Mock (1975) considered Glu-270 a proton donor in the carboxypeptidase A mechanism, but his mechanism does not favor a Glu-270/zinc-promoted water molecule as the hydrolytic nucleophile. Schepartz and Breslow (1987) observed that Glu-270 may mediate an additional proton transfer in the generation of the Pi product carboxylate. 

Fig. 34. Glu-72- Zn interactions in native carboxypeptidase A and in carboxypep-tidase A-inhibitor complexes (inhibitors have been reviewed by Christianson and Lipscomb, 1989). When substrates or inhibitors bind to the enzyme active site and interact with the zinc ion, the interaction of the metal with Glu-72 tends from bidentate toward uniden-tate coordination. The flexibility of protein-zinc coordination may be an important aspect of catalysis in this system, and the Glu-72->Zn - coordination stereochemistry observed here is consistent with the stereochemical analysis of carboxylate-zinc interactions from the Cambridge Structural Database (Carrell et al., 1988 see Fig. 4).

The metalloproteases include both exopeptidases (e.g., angiotensin-converting enzyme, aminopeptidase-M, and carboxypeptidase-A) and endopeptidases (e.g.,... 

In both carboxypeptidase A and thermolysin the active site Zn2+ is chelated by two imidazole groups and a glutamate side chain (Fig. 12-16). In carboxypeptidase A, Arg 145, Tyr 248, and perhaps Arg 127 form hydrogen bonds to the substrate. A water molecule is also bound to the Zn2+ ion. The presence of the positively charged side chain of Arg 145 and of a hydro-phobic pocket accounts for the preference of the enzyme for C-terminal amino acids with bulky, nonpolar side chains. The Zn2+ in thermolysin is also bound to two imidazole groups and that in D-alanyl-D-alanyl carboxypeptidase to three. 

Figure 16.6 The postulated productively bound complex of carboxypeptidase A and a polypeptide substrate. [Courtesy of W. N. Lipscomb.]...

The COOH-terminal amino acid of a peptide or protein may be analyzed by either chemical or enzymatic methods. The chemical methods are similar to the procedures for NH2-terminal analysis. COOH-terminal amino acids are identified by hydrazinolysis or are reduced to amino alcohols by lithium borohydride. The modified amino acids are released by acid hydrolysis and identified by chromatography. Both of these chemical methods are difficult, and clear-cut results are not readily obtained. The method of choice is peptide hydrolysis catalyzed by carboxypeptidases A and B. These two enzymes catalyze the hydrolysis of amide bonds at the COOH-terminal end of a peptide (Equation E2.3), since carboxypeptidase action requires the presence of a free a-carboxyl group in the substrate. 

In a recent paper (25), Bailey and Boulter have presented evidence for a single N-terminal methionine residue per (a suggested subunit of) 75,000 daltons. A single C-terminal sequence, —Tyr-Leu-Phe, was found using carboxypeptidases A and B and hydrazinolysis. Asparagine has also been reported as the N-terminal amino acid (59). 

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